An#bio#co(resistenza:0basi0microbiologiche,0 implicazioni ...... · uncover the molecular origins...

Gabriele Casadei – CEVA 2013

An#bio#co-‐resistenza: basi microbiologiche, implicazioni (e possibili risposte)


An#bio#co-‐Resistenza

Capacita’ di un ba>erio di resistere all’azione di una tra>amento an#bio#co

JAMA. 2012;308(18):1934-1934

Gabriele Casadei -‐ Cremona 10.07.2013


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Come funzionano gli an#bio#ci


Come funzionano gli an#bio#ci

Brock Biology of Microorganisms 13th Ed


1)  impermeabilizzazione della membrana alla penetrazione dell'an5bio5co; 2)  modificazione del target; 3)  mol5plicazione del target; 4)  vie metaboliche alterna5ve. 5)  inaFvazione dell'an5bio5co; 6)  efflusso dell'an5bio5co;

An#bio#co-‐Resistenza: Strategie generali

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An#bio#co-‐Resistenza: Membrana Impremeabile

Resistenza solitamente “Innata”/cos#tu#va

GLYCOPEPTIDI GLYCOPEPTIDI

vancomycin, teicoplanin, Telavancin, Bleomycin, Ramoplanin Avoparcina


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An#bio#co-‐Resistenza: Modificazione del Target

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An#bio#co-‐Resistenza: Modificazione del Target

Targets: DNA Girasi: srotola la doppia elica DNA Topoisomersasi: man5ene separa5 I filamen5 durante la replicazione/trascrizione

Resistenze: 1)  MUTAZIONI ANCHE PUNTIFORMI

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An#bio#co-‐Resistenza: Inacazione enzima#ca

Brock Biology of Microorganisms 13th Ed


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An#bio#co-‐Resistenza: mai un solo meccanismo singolo…


An#bio#co-‐Resistenza: Evoluzione

Qual e’ l’origine dell’An#bio#co Resistenza?

SuperbugA bacterial pathogen that is resistant to multiple antibiotics.

Although resistance has been a continuing problem since antibiotics were introduced, it is the increase in the number, diversity and range of resistant organisms that has become a huge clinical problem5. Some 50 years after the first clinical use of penicillin, which was the harbinger of a new era in infectious disease medicine, some infectious organisms such as multidrug-resistant Acinetobacter baumannii6 and Klebsiella pneumoniae7 are now virtually untreatable with currently available antibiotics.

The multiply antibiotic resistant bacteria — the so-called superbugs — are now one of the most chal-lenging problems faced by modern medicine8,9. There are at least two classes of superbugs. The first class are well-known pathogens, many of which are classified in the same genera and species as the normal human commensal flora, but which have acquired antibi-otic resistance genes and frequently have increased virulence10. Former commensal organisms such as meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and drug-resistant Escherichia coli fall into this class. The sec-ond class is opportunistic pathogens6,11, which are frequently environmental in origin and generally only infect very sick or immunocompromised patients. Opportunistic pathogens include Pseudomonas aeru-ginosa, Stenotrophomonas maltophilia, A. baumannii and Burkholderia cepacia. The bacterial opportun-ists are frequently intrinsically resistant to multiple antibiotics.

Whether of commensal or environmental origin, these resistant bacteria might have modified drug targets, reduced the influx of antibiotics, exported antibiotics through the expression of efflux pumps, or inactivated antibiotics through the expression of specific enzymes to generate a ‘perfect storm’ of resistance features that renders superbugs untreatable with currently available antibiotics.

As medical practice improves, the population of immunologically vulnerable patients — the very young, the very old, individuals who are taking immunosup-pressive drugs or those who have immunosuppressive diseases — is increasing. Unfortunately, the frequency of infection with opportunistic pathogens has also increased. This has resulted in an increased frequency of hospitalization, and hospitalization for longer peri-ods, with increased exposure to multidrug-resistant pathogens that are present in healthcare settings. In the past, most pathogens resistant to multiple antibi-otics were isolated from healthcare settings, in which antibiotic use was prevalent. Now, however, MRSA is routinely isolated from the community and, worryingly, antibiotic resistance is associated with increased viru-lence12. Penicillin-resistant Streptococcus pneumoniae (PRSP), a common pathogen of children, is also mainly community-acquired13.

These factors threaten the future continued clinical use of antibiotics. Searching for new antibiotics or mod-ifying existing chemical scaffolds will always be at best a temporary fix. What is it about antibiotic resistance, its evolution and origins that makes antibiotic resistance inevitable? Sequencing microbial genomes, cataloguing known and emerging resistance in the environment and clinics, and biochemical approaches have begun to uncover the molecular origins of antibiotic resistance.

Molecular mechanisms of antibiotic resistance An integrated network of antibiotic resistance ele-ments in bacteria provides protection against chemical threats. In bacteria, the front line of this resistance sys-tem is the cell envelope. In Gram-negative bacteria this includes the outer membrane, which is composed of an asymmetric lipopolysaccharide-phospholipid bilayer, and provides an effective physical barrier to the entry of molecules (including many antibiotics) into the cell. A cadre of outer-membrane spanning porins that facilitate the entry of small molecules into cells also passively excludes many antibiotics. In Gram-positive bacteria the absence of an outer membrane results in increased sensitivity to many antibiotics. Nonetheless, many Gram-positive bacteria, such as Mycobacteria species14, can thwart the cytotoxic effects of antibiotics through physiological defences. All bacterial genomes include genes that encode small molecule transport proteins — there are 591 such genes in E. coli, which comprise 13% of the genome15. A subset of these genes encode proteins that mediate small molecule efflux. Many of these efflux systems, such as AcrAB/TolC16, are not selective for a specific class of antibiotic, but can rid cells of various toxic compounds, thereby pro-viding innate resistance to antibiotics. Other efflux systems are highly specific and can be triggered by exposure to antibiotics. Unlike the innate resistance efflux proteins, the TetA efflux pump is selective for tetracycline antibiotics17. Expression of the tetA gene is negatively regulated by TetR, a tetracycline-sensitive gene regulator that stringently controls efflux pump expres-sion18. The evidence for the association of resistance with a loss of fitness is outlined in BOX 1.

Timeline | Antibiotic drug discovery

1908 1932 1940 1950 1960 1962 2000 2003

Protonsil (sulfonamide)

Salvarasan (arsenical)

(1940–1950) • Gramicidin (peptide)• Penicillin (!-lactam)• Neomycin (aminoglycoside)• Streptomycin (aminoglycoside)• Cephalosporin (!-lactam)

(1950–1960) • Chloramphenicol (phenylpropanoid)• Chlortetracycline (tetracycline)• Polymyxin (lipopeptide)• Erythromycin (macrolide)• Vancomycin (glycopeptide)• Virginiamycin (streptogramin)

The class of the antibiotic is shown in brackets.

Rifamycin (ansamycin)

Naladixic acid (quinolone)

Daptomycin (lipopeptide)

Linezolid (oxazolidinone)

REVIEWS

176 | MARCH 2007 | VOLUME 5 www.nature.com/reviews/micro



Nature Reviews Microbiology 5, 175-‐186 (March 2007)









1908 1932 1940 1950 1960 1962 2000 2003










REVIEWS


Aumento delle resistenze



Fenomeno indo>o??? Nature Reviews Microbiology 5, 175-‐186 (March 2007)



MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Sept. 2010, p. 417–433 Vol. 74, No. 31092-2172/10/$12.00 doi:10.1128/MMBR.00016-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Origins and Evolution of Antibiotic ResistanceJulian Davies* and Dorothy Davies

Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia,2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada

INTRODUCTION .......................................................................................................................................................417A LITTLE ANTIBIOTIC HISTORY ........................................................................................................................418SUPERBUGS AND SUPERRESISTANCE .............................................................................................................419MECHANISMS AND ORIGINS OF ANTIBIOTIC RESISTANCE ....................................................................421

Genetic Jugglery......................................................................................................................................................421Intrinsic Resistance ................................................................................................................................................422The Resistome .........................................................................................................................................................422The Subsistome .......................................................................................................................................................423Metagenomic Analyses of Environmental Samples............................................................................................423Resistance Due to Anthropogenic Activities .......................................................................................................423

GENETICS OF RESISTANCE .................................................................................................................................424Resistance Gene Transmission .............................................................................................................................425Integrons ..................................................................................................................................................................426

ECOLOGICAL ROLES OF ANTIBIOTICS AND ANTIBIOTIC RESISTANCE..............................................427The Parvome (the World of Small Molecules) ...................................................................................................428

HOW TO CONTROL OR REDUCE ANTIBIOTIC RESISTANCE DEVELOPMENT....................................428CONCLUSIONS .........................................................................................................................................................429ACKNOWLEDGMENTS ...........................................................................................................................................430REFERENCES ............................................................................................................................................................430

INTRODUCTION

The successful use of any therapeutic agent is compromisedby the potential development of tolerance or resistance to thatcompound from the time it is first employed. This is true foragents used in the treatment of bacterial, fungal, parasitic, andviral infections and for treatment of chronic diseases such ascancer and diabetes; it applies to ailments caused or sufferedby any living organisms, including humans, animals, fish,plants, insects, etc. A wide range of biochemical and physio-logical mechanisms may be responsible for resistance. In thespecific case of antimicrobial agents, the complexity of theprocesses that contribute to emergence and dissemination ofresistance cannot be overemphasized, and the lack of basicknowledge on these topics is one of the primary reasons thatthere has been so little significant achievement in the effectiveprevention and control of resistance development. Most inter-national, national, and local agencies recognize this seriousproblem. Many resolutions and recommendations have beenpropounded, and numerous reports have been written, but tono avail: the development of antibiotic resistance is relentless.

The most striking examples, and probably the most costly interms of morbidity and mortality, concern bacteria. The dis-covery of these infectious agents in the late 19th century stim-ulated the search for appropriate preventative and therapeuticregimens; however, successful treatment came only with thediscovery and introduction of antibiotics half a century later.

Antibiotics have revolutionized medicine in many respects, andcountless lives have been saved; their discovery was a turningpoint in human history. Regrettably, the use of these wonderdrugs has been accompanied by the rapid appearance of resis-tant strains. Medical pundits are now warning of a return to thepreantibiotic era; a recent database lists the existence of morethan 20,000 potential resistance genes (r genes) of nearly 400different types, predicted in the main from available bacterialgenome sequences (85). Fortunately, the number existing asfunctional resistance determinants in pathogens is muchsmaller.

Many excellent reviews describing the genetics and biochem-istry of the origins, evolution, and mechanisms of antibioticresistance have appeared over the last 60 years. Two of note inrecent times are those of Levy and Marshall (82) and White etal. (149). The goal of this short article is not to summarize sucha wealth of information but to review the situation as we see itnow (most particularly with respect to the origins and evolu-tion of resistance genes) and to provide some personal viewson the future of antibiotic therapy of infectious diseases.

Antibiotic discovery, modes of action, and mechanisms ofresistance have been productive research topics in academia(27) and, until recently, in the pharmaceutical industry. Asnatural products, they provide challenging intellectual exer-cises and surprises with respect to their chemical nature, bio-synthetic pathways, evolution, and biochemical mode of action(26, 134). The total synthesis of such natural products in thelaboratory is difficult, since these small molecules are oftenextremely complex in functionality and chirality (98). The an-tibiotic penicillin was discovered in 1928, but the completestructure of this relatively simple molecule was not revealeduntil 1949, by the X-ray crystallographic studies of Dorothy

* Corresponding author. Mailing address: Department of Micro-biology and Immunology, Life Sciences Institute, University of Brit-ish Columbia, 2350 Health Sciences Mall, Vancouver, British Co-lumbia V6T 1Z3, Canada. Phone: (604) 822-5856. Fax: (604) 822-6041. E-mail: [email protected].

417

La Resistenza e’ un fenomeno naturale! Esisteva ancora prima della scoperta degli an#bio#ci!

Antibiotics are arguably the most successful form of chemotherapy developed in the twentieth century and perhaps over the entire history of medicine. The first systematic searches for antimicrobial agents were carried out at the end of the nineteenth century in the wake of the acceptance of the ‘germ theory of disease’, which followed the conclusive experiments of researchers including Pasteur and Koch. Ehrlich’s pioneering small molecule screening approaches and the successful identification of the first anti-trypano-somal and anti-syphilitic ‘magic bullet’ drugs ushered in the modern era of antimicrobial therapy1. The first well-characterized antibacterial agents were derived from synthetic efforts, and emerged from the new science of synthetic organic chemistry that sustained the European dye industry.

However, it was the discovery of exquisitely potent, chemically diverse, and relatively non-toxic antibiotics derived from environmental bacteria and fungi, exem-plified by the penicillin family, that shifted the antimi-crobial drug-discovery paradigm from synthesizing small molecules to exploiting natural products. These discoveries paved the way for the ‘golden era’ of antibiotic discovery (1945–1960) during which most of the chemi-cal classes of antibiotics now in clinical use were first characterized (see TIMELINE). This period was followed by the extensive medicinal chemical elaboration of these chemical scaffolds over the next decade (1970–1980) to tailor these drugs to improve pharmacology and evade antibiotic resistance — the ‘golden age of antibiotic medicinal chemistry’. Relatively few antibiotic scaffolds are purely synthetic in origin. The lipopeptide daptomy-cin and the oxazolidinone linezolid were approved by

the FDA in 2003 and 2000, respectively, but daptomycin and the oxazolidinone class were both first discovered in the mid 1980s.

The pace of the discovery of new and clinically useful classes of antibiotics from the early 1960s to the present day has dramatically slowed. The reasons for this are complex and include the challenge of iden-tifying new classes of antimicrobial agents from the natural product or synthetic small molecule collec-tions available to the antibiotics industry, and a shift in focus after ~1960 in antibiotic drug development from discovery to medicinal chemical modification of existing antibiotic chemical scaffolds. Economic rea-sons for the decrease in discovery of new antibiotics include the number of many older off-patent agents that negatively influence drug pricing (antibiotics are among the cheapest drugs), the poor results of target-based drug discovery over the past decade, pressure to use new compounds sparingly to avoid resistance, and the perception that increasingly stringent criteria of regulatory agencies for new drugs are a barrier to bringing products to the market2–4.

The problem of antibiotic resistanceOne of the most important and sustained driving forces for antibiotic discovery over the past half century has been antibiotic resistance. Simply put, resistance is the continued growth of microorganisms in the presence of cytotoxic concentrations of antibiotics. Resistance is therefore relative, and as a result is defined operationally. In infectious disease clinical practice, antibiotic resist-ance that results in clinical failure is governed by the bioavailability of the antibiotics.

Antimicrobial Research Centre, Department of Biochemistry and Biomedical Sciences, DeGroote School of Medicine, McMaster University, 1200 Main Street West Hamilton, Ontario, L8N 3Z5, Canada. e-mail: [email protected]:10.1038/nrmicro1614

The antibiotic resistome: the nexus of chemical and genetic diversityGerard D. Wright

Abstract | Over the millennia, microorganisms have evolved evasion strategies to overcome a myriad of chemical and environmental challenges, including antimicrobial drugs. Even before the first clinical use of antibiotics more than 60 years ago, resistant organisms had been isolated. Moreover, the potential problem of the widespread distribution of antibiotic resistant bacteria was recognized by scientists and healthcare specialists from the initial use of these drugs. Why is resistance inevitable and where does it come from? Understanding the molecular diversity that underlies resistance will inform our use of these drugs and guide efforts to develop new efficacious antibiotics.

NATURE REVIEWS | MICROBIOLOGY VOLUME 5 | MARCH 2007 | 175

REVIEWS

In natura esistono MOLTI piu’ geni di resistenza rispe>o a quelli che solitamente si cercano nei patogeni (~24,000)


An#bio#co-‐Resistenza: Origine

Evento mutazionale naturale e raro!

E. coli = 1:1000


An#bio#co-‐Resistenza: Origine

Evento mutazionale naturale e raro: Tasso di mutazione spontanea

Epidem. Inf. (1987) 98, 361-368 361Printed in Great Britain

Antibiotic sensitivity and mutation rates to antibiotic resistance inMycoplasma mycoides ssp. mycoides

BY D. H. LEE, R. J. MILES* AND J. R. M. INALDepartment of Microbiology, Kings College, University of London,

Campden Hill Road, London W8 7AH

(Accepted 30 December 1986).

SUMMARY

The antibiotic resistance of Mycoplasma mycoides ssp. mycoides strain T, wasinvestigated. This strain was resistant to high levels ( > 100 jug ml- 1) of rifampicinand nalidixic acid. It was sensitive to streptomycin, spectinomycin and novo-biocin; however, single step mutants with high levels ofresistance ( > 100 ,tg ml-')were readily isolated. With erythromycin and tylosin for which the minimuminhibitory concentration (MIC) for the parent strain was < 01 jug ml-', mutantsresistant to > 100 jug ml-' were obtained in two and three steps respectively. TheMIC of tetracycline in single step resistant mutants (0-6,g ml-') was tenfoldhigher than the parent strain, but could not be increased further. There was onlya twofold increase in resistance to chloramphenicol in single step mutants. Thefrequency of resistant mutants varied with the antibiotic and was between4 x 10-6 and 2 x 10-8. The mutation rate to antibiotic resistance to streptomycin,spectinomycin, novobiocin, erythromycin and tylosin was between 3 x 10-8 and5 x 10-9 per cell per generation. There was a fivefold decrease in mutation rate toresistance to 60 jig ml-l streptomycin compared to that to 20 ,ug ml-.

INTRODUCTIONThere is relatively little information in the literature concerning antibiotic

resistance in mycoplasmas. In particular, there are no reports on mutation rates,and few on frequencies of resistance in cell populations.

In the work reported in this paper the antibiotic resistance of Mycoplasmamycoides ssp. mycoides, the causative agent of contagious bovine pleuropneumonia(CBPP), was investigated. Minimum inhibitory concentration (MIC) values forstrain T, and for antibiotic-resistant strains derived from it, and the frequenciesof resistant organisms were determined; mutation rates to resistance to someantibiotics were also determined using a fluctuation test (Luria & Delbriik, 1943).In addition to providing information on antibiotic resistance, it was envisagedthat the study might indicate the potential usefulness of antibiotic resistantmarkers in genetic studies. We have previously reported the isolation of substrate-negative mutants of M. mycoides (Lee, Miles & Beezer, 1986).

* Author for correspondence.

Antibiotic resistance in Mycoplasma mycoidesTable 2. Mutation rates to antibiotic resistance

The rates shown are representative values from individual experiments. In replicate experiments(observed mutation rates were always found to fall within the 95% confidence limits.)

Concentration Mutation rate (x 10-9) per cell perAntibiotic (/sg ml-l) generation with 95% confidence limitsStreptomycin 20 29 + 75Streptomycin 60 64+ 3X6Spectinomycin 60 96 + 4-8Tylosin 0 04 59 + 3 0Erythromycin 0-16 5-2+228Novobiocin 20 14 + 5-1

biotic free broth (100 ,ul inoculum per 9 ml of broth) for a minimum of three times.In all cases no reduction in antibiotic resistance was observed.

Frequencies of antibiotic-resistant mutantsThe frequency with which single-step antibiotic-resistant mutants were isolated

from the parent strain at 3 2 x MIC was between 4 x 10-6 and 2 x o0-8 (Table 1),and multi-step mutants resistant to erythromycin and tylosin were isolated fromsingle step mutants with frequencies of 10-7 to 10-8. At 1 x MIC of streptomycin,spectinomycin, nalidixic acid, erythromycin and tetracycline, the frequencies withwhich colonies appeared depended strongly upon inoculum size, e.g. with strep-tomycin (10 jug ml-'), an inoculum of 107 c.f.u. gave more than 400 colonieswhereas an inoculum of 106 gave none. These results were reproducible suggesting,possibly, that there is sufficient uptake of antibiotics by large populations toreduce the effective antibiotic concentration below the MIC. This effect was notobserved at 3 2 x MIC where the number of colonies appearing on plates wasproportional to the number of c.f.u. added.

Mutation rates to antibiotic resistanceMutation rates for single-step resistance to streptomycin, spectinomycin, tylo-

sin, erythromycin and novobiocin by the parent strain are given in Table 2. Theantibiotic concentration used to assess the presence of mutants was the MIC fornovobiocin and tylosin; however, 2-6 times the MIC was used for the otherantibiotics, for which growth at the MIC was dependent upon inoculum size.The mutation rate to resistance to 20 jug ml-' streptomycin was significantly

higher than that to 60 ,ug ml-1. Variation of mutation rate with streptomycinconcentration has previously been reported to occur in Salmonella typhi andPseudomonas aeruginosa, but not in Escherichia coli and Shigella sp. (Alexanderet al. 1950).

DISCUSSION

Of the nine antibiotics studied tylosin, tetracycline and erythromycin were themost active, with MIC values < 01 jug ml-' for M. mycoides strain T1. The MICvalues for streptomycin and tylosin (10 and 0 04 ,tg ml-' respectively) were similarto those previously reported for M. mycoides strain V5 (8 and 0-07 g ml-1;Turner, 1960; Hudson & Etheridge, 1965), though the value for tetracycline was

365

In generale gli ordini di grandezza vanno da 10-‐7 a 10-‐9


An#bio#co

Mutante spontaneo

Pressione di selezione: Il vecchio credo…

Livello di resistenza

Nel 1945 durante il suo discorso per il Nobel Prize, Alexander Fleming mise in guardia I medici: ‘‘if you use penicillin, use enough. Only by killing all bacteria in an infec#on, he asserted, could drug resistance be prevented”.


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Aggressive Chemotherapy and the Selection of DrugResistant PathogensSilvie Huijben1¤a*, Andrew S. Bell1, Derek G. Sim1, Danielle Tomasello1¤b, Nicole Mideo1, Troy Day2,

Andrew F. Read1,3*

1Center for Infectious Disease Dynamics, Departments of Biology and Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America,

2Departments of Mathematics, Statistics and Biology, Jeffery Hall, Queen’s University, Kingston, Ontario, Canada, 3 Fogarty International Center, National Institutes of

Health, Bethesda, Maryland, United States of America

Abstract

Drug resistant pathogens are one of the key public health challenges of the 21st century. There is a widespread belief thatresistance is best managed by using drugs to rapidly eliminate target pathogens from patients so as to minimize theprobability that pathogens acquire resistance de novo. Yet strong drug pressure imposes intense selection in favor ofresistance through alleviation of competition with wild-type populations. Aggressive chemotherapy thus generatesopposing evolutionary forces which together determine the rate of drug resistance emergence. Identifying treatmentregimens which best retard resistance evolution while maximizing health gains and minimizing disease transmissionrequires empirical analysis of resistance evolution in vivo in conjunction with measures of clinical outcomes andinfectiousness. Using rodent malaria in laboratory mice, we found that less aggressive chemotherapeutic regimenssubstantially reduced the probability of onward transmission of resistance (by .150-fold), without compromising healthoutcomes. Our experiments suggest that there may be cases where resistance evolution can be managed more effectivelywith treatment regimens other than those which reduce pathogen burdens as fast as possible.

Citation: Huijben S, Bell AS, Sim DG, Tomasello D, Mideo N, et al. (2013) Aggressive Chemotherapy and the Selection of Drug Resistant Pathogens. PLoSPathog 9(9): e1003578. doi:10.1371/journal.ppat.1003578

Editor: L. David Sibley, Washington University School of Medicine, United States of America

Received March 18, 2013; Accepted July 10, 2013; Published September 12, 2013

Copyright: ! 2013 Huijben et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the National Institute of General Medical Sciences (R01GM089932). The funders had no role in study design, data collectionand analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected] (SH); [email protected] (AFR)

¤a Current address: CRESIB - Barcelona Centre for International Health Research, Barcelona, Spain.¤b Current address: Neuroscience Program, The State University of New York at Buffalo, Buffalo, New York, United States of America.

Introduction

At the end of his 1945 Nobel Prize lecture, Alexander Flemingwarned of the dangers of drug resistance and proclaimed that ‘‘ifyou use penicillin, use enough’’ [1]. Only by killing all bacteria inan infection, he asserted, could drug resistance be prevented.Today, this remains standard thinking [2–12]. The philosophy isthat aggressive chemotherapy minimizes the probability thatpathogens acquire resistance by de novo mutations or lateraltransfer of genetic material: dead bugs can’t evolve. It is whyphysicians frequently exhort patients to finish drug courses evenafter they no longer feel sick [13].Yet the rate of adaptive evolution is determined by the

available genetic variation and the strength of selection [14].This means that the rate of spread of resistance alleles within ahost or within a host population is a function not only of the rateat which resistance alleles arise but also of the strength ofselection acting on them. This selection acts both within the hostwhen a genetic event conferring resistance first occurs, and thensubsequently as resistance spreads in a host population. And allelse being equal, increasing drug pressure will increase thestrength of selection. Consequently, aggressive treatment regi-mens - those aimed at eliminating all pathogens as fast aspossible by, for instance, sufficiently high drug dose or long

treatment duration - are a double-edged sword for resistancemanagement [15]. Aggressive chemotherapy can retard theevolution of resistance by reducing pathogen population sizesand hence the chances of high-level resistance arising de novo.But in an infection which already contains drug-resistantpathogens, either by de novo mutation, lateral transfer, or bytransmission from other hosts, aggressive chemotherapy willrapidly eliminate drug-susceptible competitors, thus poweringthe very evolution it is designed to inhibit.Quite how these opposing evolutionary forces combine to affect

the rate of resistance evolution in any particular host-parasitesystem is unclear. Yet without understanding that, it is impossibleto determine whether Fleming’s rule (or others, like ‘hit hard andhit fast’ [7]) are in fact sound resistance management strategies.This is particularly critical where toxicity or cost considerationsplace upper bounds on how much drug pressure can be applied, orwhere high level resistance is frequently acquired, either de novo orfrom other people. The question then becomes: among the widerange of drug doses, inter-dose intervals and treatment durationsthat can achieve the required clinical outcomes, which patienttreatment regimen best retards the evolution of resistance? Herewe present the first empirical data that shows that these need notbe treatment regimens which remove susceptible pathogens as fastas possible.

PLOS Pathogens | www.plospathogens.org 1 September 2013 | Volume 9 | Issue 9 | e1003578

ATTENTI A DOSAGGI/TEMPI

2013


An#bio#co

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Pressione di selezione!


1) La presenza di an#bio#co seleziona i ba>eri piu’ resisten#

2) La presenza con#nua di an#bio#co consolida la resistenza


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Fonte CDC 2012

CHAPTER 26 • Microbial Growth Control 769

Figure 26.14 Annual worldwide production and use of antibiotics.Each year an estimated 10,000 metric tons of antimicrobial agents aremanufactured worldwide. The !-lactam antibiotics include cepha-halosporins (30%), penicillins (7%), and other !-lactams (15%). “Others”includes tetracyclines, aminoglycosides, and all other antimicrobial drugs.

52%ß-Lactam

4%Other

20%Macrolide

24%Fluoroquinolones

OH

OH

OH

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Figure 26.15 Salvarsan. This arsenic-containing compound was oneof the first useful antimicrobial agents. It was used to treat syphilis.

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Figure 26.16 Sulfa drugs. (a) The simplest sulfa drug, sulfanilamide.(b) Sulfanilamide is an analog of p-aminobenzoic acid, a precursor of (c) folic acid, a growth factor.

resulted from the large-scale screening of chemicals for activityagainst streptococcal infections in experimental animals.

Sulfanilamide, the simplest sulfa drug, is an analog of p-aminobenzoic acid, which is itself a part of the vitamin folicacid, a nucleic acid precursor (Figure 26.16). Sulfanilamideblocks the synthesis of folic acid, thereby inhibiting nucleic acidsynthesis. Sulfanilamide is selectively toxic in bacteria becausebacteria synthesize their own folic acid, whereas most animalsobtain folic acid from their diet. Initially, sulfa drugs were widelyused for treatment of streptococcal infections ( Section 33.2).However, resistance to sulfonamides has been increasing becausemany formerly susceptible pathogens have developed an abilityto take up folic acid from their environment. Antimicrobial ther-apy with sulfamethoxazole (a sulfa drug) plus trimethoprim, arelated folic acid synthesis competitor, is still effective in manyinstances because the drug combination produces sequentialblocking of the folic acid synthesis pathway. Resistance to thisdrug combination requires that two mutations in genes of thesame pathway occur, a relatively rare event.

IsoniazidIsoniazid ( Figure 33.11) is an important growth factor analogwith a very narrow spectrum of activity (Figure 26.13). Effectiveonly against Mycobacterium, isoniazid interferes with the syn-thesis of mycolic acid, a mycobacterial cell wall component.A nicotinamide (vitamin) analog, isoniazid is the most effectivesingle drug used for control and treatment of tuberculosis ( Section 33.4).

Nucleic Acid Base AnalogsAnalogs of nucleic acid bases formed by addition of a fluorine orbromine atom are shown in Figure 26.17. Fluorine is a relativelysmall atom and does not alter the overall shape of the nucleicacid base, but changes the chemical properties such that thecompound does not function in cell metabolism, thereby block-ing nucleic acid synthesis. Examples include fluorouracil, an ana-log of uracil, and bromouracil, an analog of thymine. Growth

UN

IT 8

the authentic growth factor prevent the analog from functioningin the cell, disrupting cell metabolism. Analogs are known formany important biomolecules, including vitamins, amino acids,purines and pyrimidines, and other compounds. We begin byconsidering antibacterial growth factor analogs. Growth factoranalogs effective for the treatment of viral and fungal infectionswill be discussed in Sections 26.10 and 26.11.

Sulfa DrugsSulfa drugs, discovered by Gerhard Domagk in the 1930s, werethe first widely used growth factor analogs that specifically inhib-ited the growth of bacteria. The discovery of the first sulfa drug


Selection of Resistant Bacteria at Very Low AntibioticConcentrationsErik Gullberg1", Sha Cao1", Otto G. Berg2, Carolina Ilback1, Linus Sandegren1, Diarmaid Hughes1, Dan I.

Andersson1*

1Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden, 2Department of Molecular Evolution, Uppsala University, Uppsala,

Sweden

Abstract

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability totreat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically,but what role the much lower antibiotic concentrations present in many environments plays in selection remains largelyunclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs atextremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to severalhundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, evenwhen present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations ofantibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptiblepopulation. These results add another dimension to the evolution of resistance and suggest that the low antibioticconcentrations found in many natural environments are important for enrichment and maintenance of resistance inbacterial populations.

Citation: Gullberg E, Cao S, Berg OG, Ilback C, Sandegren L, et al. (2011) Selection of Resistant Bacteria at Very Low Antibiotic Concentrations. PLoS Pathog 7(7):e1002158. doi:10.1371/journal.ppat.1002158

Editor: Marc Lipsitch, Harvard School of Public Health, United States of America

Received December 21, 2010; Accepted May 25, 2011; Published July 21, 2011

Copyright: ! 2011 Gullberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grants from Formas, Swedish Strategic Research Foundation (SSF), the Swedish Research Council and EU 7th FrameworkProgram (project PAR) to DIA and DH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


* E-mail: [email protected]

" Shared first authorship.

Introduction

Antibiotics represent one of mankind’s most important medicalinventions but during the last decades the continuing rapiddevelopment of antibiotic resistance has emerged as one of themost serious health care problems, both in community andhospital settings [1,2,3]. Whereas some resistance-conferring geneswere most likely originally selected to serve metabolic functionsand/or for signal trafficking or protection against competingantibiotic-producing bacteria [4], the recent worldwide enrich-ment and spread of highly resistant pathogenic bacteria in themicro-biosphere has largely been driven by human activities,including the extensive use and misuse of antibiotics in human andveterinary medicine and in agriculture [2,3,5,6,7]. While it isevident that the high concentrations of antibiotics used therapeu-tically can select for resistant mutants, it still remains unclear howimportant the low antibiotic concentrations that due to anthro-pogenic input pollute natural (e.g. aquatic or soil) environments[8,9,10], that are produced naturally by antibiotic-producingmicro-organisms or that are present in certain human/animalbody compartments during therapeutic or growth promotion use,are for the selection and enrichment of resistant mutants. Inpharmacodynamic models it is generally assumed that selection ofresistant bacteria only occurs at concentrations between theminimal inhibitory concentration (MIC) of the susceptible wildtype population (MICsusc) and that of the resistant bacteria

(MICres) [11,12] (mutant selective window hypothesis, see Fig. 1A)and that concentrations below the MICsusc will not inhibit growthof the susceptible bacteria and therefore not be selective. Earlierstudies on selection with small differences in bacterial susceptibilityto antibiotics show that selection can efficiently act on minutedifferences to select for resistance [13,14,15]. Furthermore, usingan elegant color-based assay a recent study has shown qualitativelythat levels of antibiotics below the MIC can enrich for resistantbacteria [16]. Here, we further explore the mutant selectivewindow assumption and as outlined schematically in Fig. 1A, weexamine for two bacterial species and three antibiotics how farbelow the MICsusc pre-existing and de novo generated resistantmutants can be selectively enriched because of minute reductionsin the growth rate of their susceptible counterparts.To determine if exposure to very low antibiotic concentrations

(,,MICsusc) can result in enrichment for resistant mutants, weused several well-defined mutants of Escherichia coli and Salmonellaenterica (Var. Typhimurium LT2) (Table S1 in Text S1) and threedifferent classes of antibiotics with high importance to human andveterinary medicine (tetracyclines, fluoroquinolones and amino-glycosides). The resistance markers used were Tn10dTet (conferstetracycline resistance), gyrA (S83L and D87N), DmarR, and DacrRmutations (confer ciprofloxacin resistance) and rpsL (K42R)(confers streptomycin resistance), all of which are found in clinicalisolates of several different bacterial species. Using highly sensitivecompetition experiments between isogenic pairs of susceptible and

PLoS Pathogens | www.plospathogens.org 1 July 2011 | Volume 7 | Issue 7 | e1002158

Pressione di selezione…

Selection of Resistant Bacteria at Very Low AntibioticConcentrationsErik Gullberg1", Sha Cao1", Otto G. Berg2, Carolina Ilback1, Linus Sandegren1, Diarmaid Hughes1, Dan I.

Andersson1*

1Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden, 2Department of Molecular Evolution, Uppsala University, Uppsala,

Sweden

Abstract

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability totreat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically,but what role the much lower antibiotic concentrations present in many environments plays in selection remains largelyunclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs atextremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to severalhundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, evenwhen present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations ofantibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptiblepopulation. These results add another dimension to the evolution of resistance and suggest that the low antibioticconcentrations found in many natural environments are important for enrichment and maintenance of resistance inbacterial populations.

Citation: Gullberg E, Cao S, Berg OG, Ilback C, Sandegren L, et al. (2011) Selection of Resistant Bacteria at Very Low Antibiotic Concentrations. PLoS Pathog 7(7):e1002158. doi:10.1371/journal.ppat.1002158

Editor: Marc Lipsitch, Harvard School of Public Health, United States of America

Received December 21, 2010; Accepted May 25, 2011; Published July 21, 2011

Copyright: ! 2011 Gullberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grants from Formas, Swedish Strategic Research Foundation (SSF), the Swedish Research Council and EU 7th FrameworkProgram (project PAR) to DIA and DH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


* E-mail: [email protected]

" Shared first authorship.

Introduction

Antibiotics represent one of mankind’s most important medicalinventions but during the last decades the continuing rapiddevelopment of antibiotic resistance has emerged as one of themost serious health care problems, both in community andhospital settings [1,2,3]. Whereas some resistance-conferring geneswere most likely originally selected to serve metabolic functionsand/or for signal trafficking or protection against competingantibiotic-producing bacteria [4], the recent worldwide enrich-ment and spread of highly resistant pathogenic bacteria in themicro-biosphere has largely been driven by human activities,including the extensive use and misuse of antibiotics in human andveterinary medicine and in agriculture [2,3,5,6,7]. While it isevident that the high concentrations of antibiotics used therapeu-tically can select for resistant mutants, it still remains unclear howimportant the low antibiotic concentrations that due to anthro-pogenic input pollute natural (e.g. aquatic or soil) environments[8,9,10], that are produced naturally by antibiotic-producingmicro-organisms or that are present in certain human/animalbody compartments during therapeutic or growth promotion use,are for the selection and enrichment of resistant mutants. Inpharmacodynamic models it is generally assumed that selection ofresistant bacteria only occurs at concentrations between theminimal inhibitory concentration (MIC) of the susceptible wildtype population (MICsusc) and that of the resistant bacteria

(MICres) [11,12] (mutant selective window hypothesis, see Fig. 1A)and that concentrations below the MICsusc will not inhibit growthof the susceptible bacteria and therefore not be selective. Earlierstudies on selection with small differences in bacterial susceptibilityto antibiotics show that selection can efficiently act on minutedifferences to select for resistance [13,14,15]. Furthermore, usingan elegant color-based assay a recent study has shown qualitativelythat levels of antibiotics below the MIC can enrich for resistantbacteria [16]. Here, we further explore the mutant selectivewindow assumption and as outlined schematically in Fig. 1A, weexamine for two bacterial species and three antibiotics how farbelow the MICsusc pre-existing and de novo generated resistantmutants can be selectively enriched because of minute reductionsin the growth rate of their susceptible counterparts.To determine if exposure to very low antibiotic concentrations

(,,MICsusc) can result in enrichment for resistant mutants, weused several well-defined mutants of Escherichia coli and Salmonellaenterica (Var. Typhimurium LT2) (Table S1 in Text S1) and threedifferent classes of antibiotics with high importance to human andveterinary medicine (tetracyclines, fluoroquinolones and amino-glycosides). The resistance markers used were Tn10dTet (conferstetracycline resistance), gyrA (S83L and D87N), DmarR, and DacrRmutations (confer ciprofloxacin resistance) and rpsL (K42R)(confers streptomycin resistance), all of which are found in clinicalisolates of several different bacterial species. Using highly sensitivecompetition experiments between isogenic pairs of susceptible and

PLoS Pathogens | www.plospathogens.org 1 July 2011 | Volume 7 | Issue 7 | e1002158


Pressione di selezione…

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Ecology of Antibiotic Resistance Genes 13

But how likely are such exchanges to occur, especially broad host range transfers between members of different species and genera? This is the type of transfer that could be most problematic because it would allow resistance genes to move into bacteria capable of causing human disease. In trying to answer this question, atten-tion has focused on conjugative gene transfer because this is the type of transfer known to be capable of crossing genus and phylum lines [20]. Initially, however, the focus was somewhat larger because early studies sought examples in which the same gene, with “same” de! ned as DNA sequence identity of more than 95%, was found in two very distantly related species of bacteria. That is, the only criterion was evi-dence that some sort of DNA transfer had occurred, without specifying the mecha-nism. The 95% cutoff was arbitrary but was motivated by the need to eliminate the possibility of convergent evolution. In convergent evolution, the same amino acid sequence might arise by selection from two different genes. Since two genes can dif-fer by as much as 20% at the DNA sequence level and still have the same amino acid sequence, the requirement for 95% or higher DNA sequence identity seemed to be a good way to restrict attention to recent horizontal transfers of resistance genes.

In fact, the cutoff could have been 98%, because it proved all too easy to ! nd resistance genes in different genera and species that were 98% to 100% identical at the DNA sequence level. Some examples are shown in Figure 2.2, where the resistance gene designation is shown inside the oval at the center and the names of Gram-positive and Gram-negative bacterial species found to have that gene are shown on either side of the oval. What is striking about this ! gure is that not only has the same gene been found in widely divergent species, but also in species commonly found in different locations. That is, the same genes were found not only in human colonic bacteria, but also in bacteria from other sites, such as soil, the intestinal tracts of

FIGURE 2.2 Example of genes with more than 95% sequence identity that have been found in distantly related bacteria from different sites.

9190_C002.indd 139190_C002.indd 13 10/26/2007 7:27:52 PM10/26/2007 7:27:52 PM

Bacterial resistance to anAmicrobials 2nd ediAon



12 Bacterial Resistance to Antimicrobials

bacterial cells located in a natural setting. Thus, the pattern of resistance gene distri-bution could be as important, if not more so, than the distribution of resistant strains of a particular species. This is especially true if resistance genes from one species can move to another species. Even if a newly acquired resistance gene is not expressed initially in a bacterial host, selective pressures imposed by the widespread clinical and agricultural use of antibiotics could select for promoter or codon usage muta-tions that allow the resistance gene to be expressed [6,7].

The importance of understanding the ! ow of resistance genes became particu-larly evident in discussions of possible impacts of agricultural use of antibiotics. In this case, initial attention focused on Salmonella and Campylobacter spp., types of bacteria that could cause human disease. Attention soon expanded, however, to include a broader question. Was it possible that even non-pathogenic bacteria, moving through the food supply from farm to the consumer, could transfer resistance genes to human intestinal bacteria [8–10]? Since human intestinal bacteria are a common cause of post-surgical infections [11,12], increased resistance due to acquisition of genes from swallowed bacteria passing through the intestinal tract could indeed have a direct impact on human health [13,14].

Assertions such as this prompted an old idea, called the “reservoir hypothesis” to resurface [15–17]. The reservoir hypothesis as it applies to human colonic bacteria is illustrated in Figure 2.1, but similar sorts of gene ! ows could occur almost any-where in nature. According to the reservoir hypothesis, commensal bacteria in the colon, including those that could act as opportunistic pathogens and those that were truly non-pathogenic, exchange DNA with one another. They can also acquire DNA from or donate DNA to swallowed bacteria that cannot colonize the human colon, but spend enough time in the colon for DNA transfer to occur [18,19].

FIGURE 2.1 The reservoir hypothesis. Bacteria in the human colon serve as “reservoirs” for resistance genes that can be acquired from ingested bacteria.

9190_C002.indd 129190_C002.indd 12 10/26/2007 7:27:51 PM10/26/2007 7:27:51 PM

Bacterial resistance to anAmicrobials 2nd ediAon

An5bio5co-‐resistenza si propaga per il propagarsi di geni di resistenza, non di ba_eri resisten5…. Il DNA e’ uno dei materiali piu’ resisten5 in natura

I geni di resistenza una volta seleziona# persistono

nell’ambiente per anni!!!

Ba>eri-‐Indicatori


QUINDI CHE FARE?



An#bio#co-‐Resistenza L’unico modo per sconfiggerla e’ trovare nuove

molecole con meccanismi d’azione nuovi.









1908 1932 1940 1950 1960 1962 2000 2003










REVIEWS


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!"#"$%&'()*&++,)-,+.%&)DIPARTIMENTO DELLA SANITÁ PUBBLICA VETERINARIA, DELLA SICUREZZA

ALIMENTARE E DEGLI ORGANI COLLEGIALI PER LA TUTELA DELLA SALUTE

DIREZIONE GENERALE DELLA SANITÁ ANIMALE E DEI FARMACI VETERINARI

UFFICIO IV ex DGSA - Medicinali veterinari e dispositivi medici ad uso veterinario

!!!!!!

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Aggiornato al 27/02/13


USO RESPONSABILE DEL FARMACO

laboratorio e esperienza maturata sul campo) che la malattia da trattare è di origine batterica, cercando di identificare quanto più precisamente possibile l’agente batterico causa della malattia.

6. Quando possibile, dovrebbe sempre utilizzare i prodotti registrati per malattie infettive specifiche. In assenza di prodotti simili, al fine di scegliere l’antibiotico attivo contro un determinato microrganismo in grado di raggiungere la concentrazione efficace negli organi bersaglio, la scelta dei prodotti e delle vie di somministrazione dovrebbe essere basata - su dati di laboratorio e sulle indicazioni fornite nel foglietto illustrativo, nonché - sulle informazioni disponibili aggiornate in relazione alla farmacocinetica e farmacodinamica nella specie animale oggetto di terapia.

7. L’uso locale dell’antibiotico deve essere generalmente preferito a quello sistemico ogni qual volta sia possibile, salvo per i prodotti con questa indicazione specifica.

8. Le combinazioni empiriche di antibiotici o la modifica della posologia e della durata del trattamento rispetto a quanto riportata nel foglietto illustrativo dovrebbero essere evitati a meno che ciò non risulti essere l’ultima opzione terapeutica per salvare la vita di un animale da affezione o di particolare pregio. In tal caso qualsiasi protocollo utilizzato dovrà essere supportato da adeguata documentazione scientifica.

9. I protocolli chirurgici dovrebbero enfatizzare l’utilizzo di rigide procedure di asepsi in luogo della profilassi medica basata sull’impiego degli antibiotici.

10. L’eventuale mancata risposta clinica a un trattamento terapeutico deve essere immediatamente comunicata all’Autorità Competente (14-15-16), secondo quanto prescritto dalla normativa vigente ( vedi capitolo sulla farmacovigilanza veterinaria ) .

PRINCIPI PER UN USO RESPONSABILE DEGLI ANTIBIOTICI NEGLI ANIMALI DA

REDDITO

1. Diagnosi corretta 2. Farmacocinetica nota

3. Stato di immunocompetenza noto 4. Scelta dell’antibiotico appropriato

5. Corretta posologia 6. Verifica dei risultati

19


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<Pressione di Selezione (Uso massiccio, umano e veterinario, di an#bi#ci)


Emerging Infec,ous Diseases • www.cdc.gov/eid • Vol. 16, No. 1, January 2010


Ceriofur nuovamente acvo



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Conoscere i Nemici!


Conoscere i nemici: Conoscere “IL NORMALE”


Non costa molto fare un piano di sorveglianza interno all’allevamento!

COME? QUANDO? COSA?

Conoscere il Nemico: Conoscere “IL Patologico”


FARE DIAGNOSI!

1)  Prelievo 2)  Inizio Terapia 3)  Valutazione terapia e da# di laboratorio


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<Pressione di Selezione


1)  Conoscere prevalenza resistenze in allevamento 2) Rotazione principi acvi 3) Migliorare le pra#che manageriali


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USO RESPONSABILE DEL FARMACO

SCEGLIERE IL GIUSTO APPROCCIO/ALLEATO

Evitare il piu’ possibile l’uso preven#vo (profilacco)

Provare ad inves#re su vaccini…. E’ ANCHE LA SCELTA DI MADRE NATURA…

Potenziare Buone Pra#che Allevamento


Grazie per l’a>enzione

JAMA. 2012;308(18):1934-1934

An#bio#co(resistenza:0basi0microbiologiche,0 implicazioni ...... · uncover the molecular origins...

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